This application claims priority to Japanese Application Serial No. 67684/2000, filed Mar. 10, 2000.
The present invention relates to a method and a device for detecting a hybridization reaction between probe biopolymers such as probe DNAs spotted on a biochip and a sample biopolymer such as fluorescence-labeled or chemiluminescence-labeled DNA.
In the fields of molecular biology and biochemistry, biopolymers such as nucleic acids and proteins from organisms are identified and/or fractionated in order to search for useful genes or to diagnose diseases. A hybridization reaction is frequently used as a pretreatment for such process, where a target molecule in a sample is hybridized to nucleic acids or proteins having known sequences. In order to process mass samples in a short time, a biochip is used whose surface is provided with a plurality of features arranged in a matrix. For example, different DNA probes are spotted and immobilized on the respective features of the biochip. Such biochip is placed into a reaction container together with sample DNA to allow the fluorescence-labeled sample DNA to hybridize with the probes immobilized on the respective features of the biochip. Thereafter, the biochip is irradiated with excitation light to measure fluorescent intensity of each feature. Based on the measured fluorescent intensities, the binding levels between the respective probes and the sample DNA are obtained and converted into desired information.
FIGS. 13A and 13B are schematic views illustrating a conventional hybridization reaction using a biochip. As shown in FIG. 13A, a sample DNA solution 114 is applied onto a DNA spot region 112 of a biochip 110 provided with a plurality of DNA spots. Then, the biochip 110 is covered with a glass cover 116. As shown in FIG. 13B, the biochip 110 is then enclosed in a sealed container 120 to be subjected to hybridization in a chamber provided with a temperature controller. The temperature inside the chamber is maintained at a predetermined temperature. Although an optimal temperature for hybridization generally varies according to each DNA spot, the temperature is set to an average optimal temperature to perform hybridization to all of the spots. Thereafter, the biochip is washed to remove the non-hybridized sample DNA, followed by reading a fluorescence from fluorescent substance labeling the sample DNA with a fluorescence reader.
Conventionally, hybridization is performed while setting the temperature to an average dissociation temperature regardless of difference among individual optimal temperatures of the respective spots on the biochip. Therefore, at a spot having an optimal temperature lower than the set average temperature, binding between the complementary DNAs cannot be maintained, lowering the hybridization efficiency and resulting in a lower detected signal intensity as compared to an intrinsic signal intensity. On the other hand, at a spot having an optimal temperature higher than the set average temperature, non-specific binding is caused between non-complementary DNAs, resulting in a higher detected signal intensity as compared to an intrinsic signal intensity. According to a hybridization detection using such a conventional biochip, hybridization is performed at a constant temperature regardless of the difference among the optical temperatures of the respective spots, and the results read with the fluorescence reader are used to compare DNA levels. Thus, the quantitation results have been questionable.
In view of such conventional problems, the present invention has objectives of providing a method and a device for detecting hybridization, which are capable of detecting and quantifying only specific bindings resulting from the hybridization.
In order to realize the above-mentioned objectives, the present invention is provided with a controller having a program capable of altering a temperature of the biochip at different time points, an excitation light source and a cooled CCD camera used for detection. A sample biopolymer is hybridized to probe biopolymers on the spots of the biochip at a low temperature. The temperature is gradually raised while supplying a washing solution. Images of an entire surface of the biochip at predetermined temperatures are taken to detect fluorescence from each spot. By performing hybridization while altering from lower temperature to higher temperature, the status of the hybridization can be confirmed. As a result, hybridizations at every single spots can be detected with high reliability without being influenced by optimal hybridization temperatures which differ by types of probe biopolymers at respective spots.
A method for detecting a hybridization reaction according to the present invention comprises the steps of: binding a sample biopolymer to a biochip having a reaction region on which a plurality of probe biopolymers are separately spotted; and detecting hybridization reactions at individual spots by raising the temperature of the biochip.
In the step of detecting the hybridization reaction, a washing solution is preferably run to the reaction region of the biochip. By running the washing solution, a sample biopolymer non-hybridized to or dissociated from the probe biopolymers can be removed from the spots, thereby reducing occurrence of noise upon detection.
Furthermore, a method for detecting a hybridization of the invention comprises the steps of: placing, in a container, a biochip having a reaction region on which a plurality of probe biopolymers are separately spotted; injecting a sample biopolymer into the container; maintaining the biochip in the container at a constant temperature; and taking images of the reaction region of the biochip at predetermined timings while running a washing solution into the container and while changing the temperature of the biochip according to a predetermined time pattern. In the step of maintaining the biochip in the container at a constant temperature, a sample biopolymer is injected to be hybridized to the probe biopolymers on the spots. While supplying the washing solution into the container, the temperature of the biochip is preferably altered according to a simple temperature-raising time pattern.
Preferably, the sample biopolymer is fluorescence-labeled, and fluorescent intensities of individual spots are analyzed based on the images. Degrees of the hybridization reactions between the sample biopolymer and the individual target biopolymers immobilized on the spots may be detected based on changes of a fluorescent intensity of each spot with time. Information of a temperature upon a rapid fall of the fluorescent intensity is acquired, so as to compare it with a dissociation temperature estimated from the molecular structure of the probe biopolymer, thereby improving reliability of detection of a hybridization reaction. When the temperature upon a rapid fall is generally equal to the estimated dissociation temperature of the probe biopolymer immobilized on the spot, it may be judged that specific hybridization took place. On the other hand, when the two temperatures are obviously different from each other, it may be judged that the sample biopolymer is bound non-specifically.
A device for detecting a hybridization reaction according to the present invention comprises: a container for accommodating a biochip having a reaction region on which a plurality of probe biopolymers are separately spotted; a temperature controller for controlling the temperature of the biochip placed in the container according to a predetermined time pattern; a unit for supplying a washing solution into the container; an image pickup unit for taking an image of the reaction region of the biochip in the container; a controller for controlling timing for taking the image with the image pickup unit; and a storage unit for storing a plurality of images taken with the image pickup unit. The temperature controller preferably controls the temperature of the biochip according to a simple temperature-raising time pattern. Preferably, the device comprises a function of displaying changes of a fluorescent intensity of a selected spot with time.